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User Manual
ValidPrime™
Control for Genomic Background
Human and Mouse
Probe protocol
Version 1.1— September 2012
For use in quantitative real-time PCR
ValidPrime™
Table of contents
Background 4
Traditional approach based on RT(-) controls 6
ValidPrime™ 7
Contents 8
Storage 9
Additionally required materials
and devices 9
Amplification protocol 10
Pipetting protocol 10
GenEx 11
Troubleshooting 12
References 13
Reorder information 13
Contact 13
License information 13
Other products from TATAA
14
(HL)-dsDNase
GenEx software
Reference Gene Panel - Human or Mouse
VisiBlue qPCR mix colorant
CelluLyser™ - for rapid and easy lysis and cDNA synthesis
TATAA Interplate Calibrator - Variation Compensation
3
Background
For accurate gene expression analysis, the measured Cq shall reflect the amount
of gene transcript present in the sample. This requires that the assay is specific
and selective for the targeted cDNA and contributions to the signal from primer
dimers, pseudogenes and genomic DNA are negligible. To test for primer dimer
formation qPCR is performed in absence of template (no template control, NTC)
and to test for genomic DNA (gDNA) background reverse transcription (RT) is
performed in the absence of reverse transcriptase (RT(-)). The NTC is sample
independent and is performed only once to validate assay performance, while
the amount of gDNA may vary and RT(-) controls are typically measured on all
samples. These controls add substantially to the cost of a qPCR study.
ValidPrime™ is an assay to test for the presence of gDNA in test samples and
when combined with a gDNA control sample, replaces all RT(-) controls.
ValidPrime™ is highly optimized and specific to a non-transcribed locus
of gDNA that is present in exactly one copy per haploid normal genome.
Therefore, ValidPrime™ measures the number of genomic copies present in a
sample and can be used for normalization of samples to cell copy number, as
endogenous control for CNV applications, and as control for gDNA background
in RTqPCR. The ValidPrime™ kit also contains a gDNA standard that can be used
to test the sensitivity of RTqPCR assays for gDNA background.
4
ValidPrime™
In expression profiling experiment the ValidPrime™ assay is added to the list of
assays and the gDNA control is added to the list of samples. From the combined
measurements with the ValidPrime™ assay and the gene of interest (GOI) assays
on all samples and on the gDNA control the genomic background contribution
to all RTqPCR measurements can be assessed. ValidPrime™ replaces the need
to perform RT(-) controls for all reactions and makes RTqPCR profiling easier
and substantially cheaper. In an expression profiling experiment based on
m samples and n assays, traditional set up requires m RT(-) reactions plus
m x n qPCR controls, while using ValidPrime™ only m + n + 1 controls are
needed (Table 1).
Samples (m)
No. of
controls
Assays (n)
1
10
24
48
96
1
2
3
11
12
25
26
49
50
97
98
10
20
12
110
21
250
35
490
59
970
107
24
48
26
264
35
600
49
1176
73
2328
121
48
96
50
528
59
1200
73
2352
97
4656
145
96
192
98
1056
107
2400
121
4704
145
9312
193
Traditional RT(-) strategy
ValidPrime™
Table 1: Total number of RT and qPCR controls needed to check for gDNA
background using traditional RT(-) approach and ValidPrime™. In an expression profiling experiment based on m samples and n assays, traditional set up
requires m RT(-) reactions plus m x n qPCR controls, while using ValidPrime™
only m + n + 1 controls are needed.
5
Traditional approach based on RT(-) controls
Presence of genomic background in RTqPCR expression profiling is
conventionally assessed by running an RT(-) control for each sample that is
analyzed by qPCR for all the GOI’s. Any signal observed in these RT(-)qPCR’s
is due to presence of contaminating DNA that is amplified by the qPCR assay
designed for GOI. A common criterion to accept the measured Cq as not being
GOI
GOI
confounded by gDNA contamination is Cq RT(-) - Cq RT(+) > 5. The estimated GOI
concentration is then accurate to at least 96.9 % (Figure 1).
Figure 1: Correlation of ∆Cq RT(-)-RT(+) with percentage of gDNA in
unknown sample
GOI
GOI
GOI
If Cq RT(-)– Cq RT(+)< 5 the measured Cq RT(-) is confounded. It can be corrected to
reflect the RNA concentration using eq. 1 (Laurell et al., 2011):
Cq
GOI
RNA
(
= − log 2 2
GOI
−Cq RT
(+)
−2
GOI
−Cq RT
(−)
)
Equation 1
GOI
GOI
Cq RT(+) and Cq RT(-) are the qPCR Cq values measured for the RT(+) and RT(-)
reactions, and Cq GOI is the Cq value that would have been obtained for the
RNA
GOI
RT(+) reaction in absence of gDNA contaminations. From Cq RNA the correct
transcript amount can be calculated.
6
ValidPrime™
ValidPrime™
Using ValidPrime™ the same test for gDNA contamination can be performed
and, if needed, the same correction for background can be made, with a much
smaller number of reactions. The sensitivities of the GOI qPCR assays for gDNA
GOI
ValidPrime
( Cq gDNA ) are tested relative to the ValidPrime™ assay ( Cq gDNA
) on the
provided gDNA standard. Well performing GOI assays that have been properly
designed to exclusively target mRNA by, for example, having intron spanning
primers shall not amplify the gDNA standard, while GOI assays that amplify
sequences present in multiple copies in the gDNA will have even lower Cq
values than the ValidPrime™ assay. All samples are then analyzed also with the
ValidPrime
ValidPrime™ assay ( Cq Sample
). The measurement setup is shown in Table 2.
Original
data
GOI 1
GOI 2
GOI 3
GOI 4
ValidPrime™
Sample 1
20.1
31.1
22.1
28.2
32.5
Sample 2
20.5
31.2
22.5
28.9
33.2
Sample 3
21.0
31.1
22.9
30.2
32.3
Sample 4
23.1
31.8
22.5
32.3
34.2
Sample 5
23.5
30.8
22.8
32.0
33.1
gDNA standard
25.8
26.9
26.7
26.0
27.0
Table 2: Experimental setup based on five samples assayed for four GOI’s
and ValidPrime™ including also the gDNA standard.
GOI
Cq RT(+) is shown in black, Cq GOI in blue, Cq ValidPrime in red, Cq ValidPrime
gDNA
gDNA
Sample
in orange.
ValidPrime
ValidPrime
GOI
From the measured Cq Sample
, Cq gDNA
and Cq gDNA expected Cq for RT(-)
GOI
controls, Cq RT(-), are calculated with Equation 2 and, as before, Equation 1 is used
to correct for the gDNA background (Table 3).
(
GOI
GOI
ValidPrime
Cq RT
− CqValidPrime
gDNA
( −) = Cq gDNA + Cq Sample
)
Equation 2
7
ValidPrime™
Gene 1
Gene 2
Gene 3
GOI
GOI
GOI
GOI
GOI
GOI
GOI
GOI
Sample 1
20.1
31.3
20.10
31.1
32.4
31.85
22.1
32.2
Sample 2
20.5
32.0
20.50
31.2
33.1
31.65
22.5
32.9
Sample 3
21.0
31.1
21.00
31.1
32.2
32.01
22.9
Sample 4
23.1
33.0
23.10
31.8
34.1
32.13
Sample 5
23.5
31.9
23.50
30.8
33.0
31.15
Gene 4
GOI
GOI
GOI
GOI
22.10
28.2
31.5
28.35
22.50
28.9
32.2
29.05
32.0
22.90
30.2
31.3
31.11
22.5
33.9
22.50
32.3
33.2
33.41
22.8
32.8
22.80
32.0
32.1
35.90
CqRT(+) CqRT(-) CqRNA CqRT(+) CqRT(-) CqRNA CqRT(+) CqRT(-) CqRNA CqRT(+) CqRT(-) CqRNA
GOI
GOI
Table 3: Measured Cq RT(+) calculated Cq
using Equation 2, and
RT(-)
GOI
calculated Cq
using Equation 1.
RNA
Contents
• A 6 months complimentary license for GenEx Enterprise - the easiest way to
correct RTqPCR data for gDNA background. GenEx is market leading software
for qPCR experimental design and data processing. Read more on page 11.
1 GenEx license per customer, for first time users only.
• Reference standard gDNA 50 µl or 100 µl, C=200 ng/µl.
We recommend using 10 ng per qPCR.
• ValidPrime™ assay primers for:
250 rxns* (250 µl of primer mix, C=10 µM per primer)
or 1000 rxns* (1000 µl of primer mix, C=10 µM per primer)
• ValidPrime™ assay probe for:
250 rxns* (125 µl of solution, C=10 µM)
or 1000 rxns* (500 µl of solution, C=10 µM)
* rxns = qPCR reactions in 25 µl, concentration = 400 nM per primer, 200 nM
probe
The ValidPrime™ assay amplifies a gDNA sequence that is present in exactly one
copy per haploid genome in a normal cell. The sequence has no transcriptional
activity and is not present in pure cDNA preparations. The assay has very high
PCR efficiency (E > 90% in tested commercial master mixes) and produces negligible amount of primer dimer products. Limit of detection (LOD) is estimated
to 4 copies of DNA (0.01 ng of DNA), limit of quantification (LOQ) is estimated
to 32 copies of DNA (0.08 ng of DNA). Probe is using FAM reporter and BHQ1
quencher.
8
ValidPrime™
Storage
The ValidPrime™ kit can be stored for 1 month at +4oC. For long term storage
-20oC is recommended. Repeated freeze-thaw cycles should be avoided. Vortex thoroughly and spin down before use.
Additionally required materials and devices
• qPCR instrumentation
The ValidPrime™ kit has been validated on:
Roche LightCycler 480, Biorad CFX, Stratagene MxPro, Rotorgene, ABI 7500
Fast and is expected to perform excellent on related instruments. The ValidPrime™ probe signal shall be measured on the instrument’s FAM channel.
• Master mix or master mix components
The ValidPrime™ kit has been validated in a large number of master mixes using
the conditions recommended by the manufacturers, including:
Master mix
Final
concentrations*
Annealing
temperature
Applied Biosystems TaqMan® Gene expression Master Mix
800 nM primer, 200 nM probe
= 61°C
Applied Biosystems TaqMan® Universal PCR Master Mix
800 nM primer, 200 nM probe
= 59°C
Applied Biosystems TaqMan® Genotyping Master Mix
800 nM primer, 200 nM probe
= 59°C
Finnzymes DyNAmo™ Flash Probe qPCR Kit
500 nM primer, 250 nM probe
≈ 60°C
Finnzymes DyNAmo™ ColorFlash Probe qPCR Kit
500 nM primer, 250 nM probe
≈ 60°C
KAPA™ PROBE FAST qPCR Kit
400 nM primer, 200 nM probe
≈ 60°C
Qiagen QuantiTect Probe PCR Kit
400 nM primer, 200 nM probe
≈ 60°C
Roche LC480 Probes master
500 nM primer, 200 nM probe
≈ 60°C
* Concentration per primer and probe in qPCR
Table 4: Recommended primer and probe concentrations, and annealing
temperature in selected commercial master mixes
• Pipettes and tips (available from www.tataa.com)
• Vortex and centrifuge
• Sample RNA/DNA
• Optionally DNase
GOI
With ValidPrime™ measured Cq RT(+) can be corrected for up to 50% gDNA background. If gDNA background is high it is recommended to reduce it by treating
9
the cDNA with double strand specific DNase that will remove specifically the
gDNA and will not degrade the cDNA. We recommend heat labile dsDNase
(HL-dsDNase), which is efficiently heat inactivated and does not digest the PCR
product either. HL-dsDNase is available from www.tataa.com.
• Optionally reference cDNA
Newly designed assays can be validated on cDNA libraries. Several cDNA libraries are available from www.tataa.com.
Amplification protocol
Use the recommended amplification protocol for your master mix. Optimal
annealing temperature of the ValidPrime™ assay is about 60°C in most master
mixes, see table 4 for the optimum conditions in selected master mixes. Either
2- or 3-step amplification protocol can be used.
Pipetting protocol
Prepare a master mix for each assay using the protocol from the manufacturer,
including the recommended concentrations of primers and probe . Prepare
for at least one extra reaction so you do not run out of master mix during the
pipetting. An NTC is recommended to test for contamination of reagents. We
recommend 10 ng of gDNA per 10 μl qPCR, which on most qPCR instruments
should produce a Cq for the ValidPrime™ assay in the range 25-30 cycles.
10
ValidPrime™
GenEx
Easiest is to use GenEx for correction of RTqPCR data for gDNA background and
for general qPCR data processing. GenEx is market leading software for qPCR
experimental design and data processing, and is supported by all leading qPCR
instrument manufacturers. It offers user friendly optimized workflow for qPCR
data pre-processing and analysis. Pre-processing includes interplate calibration, efficiency correction, various normalization options, handling of technical
replicates and missing data, normalization with paired samples and correction
for gDNA contamination using ValidPrime™. Analyses include absolute quantification, relative quantification and expression profiling. Tutorials are available
on: www.multid.se/tutorials.php and free support is offered on www.qpcrforum.com.
A 6 months complimentary license for GenEx Enterprise is included for first time
users with the ValidPrime™ kit. To get started, send an email to [email protected]
and state your order number for your ValidPrime kit together with your customer details. You will receive a key to activate your free license that can be
downloaded from www.multid.se. To purchase additional GenEx licenses or for
qPCR data analysis services, contact us on [email protected].
11
Troubleshooting
• I do not get any amplification/signal?
The instrument may not have been programmed correctly or there may be a
problem with the master mix. Establish if the problem is in the detection or the
amplification by running the samples on a gel. Run a new test using the gDNA
control with the ValidPrime™ assay provided.
• My negative controls are amplified?
Your reagents are probably contaminated.
• My samples have same/higher Cq-value than my NTC?
You have used too little cDNA. Add more cDNA and try again. The cDNA may
also be of poor quality. Check the quality of the RNA before performing cDNA
synthesis.
• My replicates are not tight?
With good quality cDNA and good pipetting technique, very high reproducibility is expected. Low amounts of cDNA can lead to higher variation. Also, low
quality cDNA can lead to differences between replicates. Check the accuracy
and reproducibility of your pipettes. It is also possible the qPCR instrument is
malperforming.
• I get positive ValidPrime™ signal even after DNAse treatment?
Often DNAse treatment does not remove all DNA and qPCR will amplify a single molecule. Usually solution based DNase treatment is more efficient than
column based DNase treatment. You may also try the HL-dsDNase from TATAA
(www.tataa.com), which has superior performance to competing dsDNases.
Usually DNase treatment reduces the gDNA background enough to be accounted for by ValidPrime™ and GenEx correction.
12
ValidPrime™
References
Henrik Laurell, Jason Iacovoni, David Svec, Anne Abot, Jean-José Maoret,
Jean-François Arnal, Mikael Kubista. Correcting RTqPCR data for genomic DNA
background. Nucleic Acids Res. 2012.
Mikael Kubista, Vendula Rusnakova, David Svec, Björn Sjögreen and Ales
Tichopad. GenEx - Data Analysis Software. In qPCR in Applied Microbiology.
Editor: Martin Filion. Horizon Press, 2012.
Reorder information
The ValidPrime™ kit can be ordered from TATAA by mail at [email protected],
from our TATAA webshop on www.tataa.com or from the local TATAA distributor
in your country.
Contact
For more information about ValidPrime™, contact us at [email protected].
License information
PCR is covered by several patents owned by Hoffman-La Roche Inc., and Hoffman-LaRoche, Ltd.
Purchase of the ValidPrimeTM kit does not include or provide a license with respect to any PCR related
patents owned by Hoffman-La Roche or others. TATAA Biocenter does not encourage or support the
unauthorised or unlicensed use of the PCR process.
13
Other products from TATAA
(HL-)dsDNase
New generation DNase that is specific to double stranded DNA and can be efficiently inactivated by heating at 55 °C. It can be added to your RT reaction to
efficiently remove any gDNA, without degrading single-stranded cDNA. It is
completely inactivated by the PCR and does not degrade the double stranded
PCR product.
GenEx software
Market leading software for qPCR analysis. GenEx provides the appropriate
tools to analyze qPCR gene expression data and to extract biologically relevant
information from the measurements.
Reference Gene Panel - Human or Mouse
The panel contains primer sets for 12 commonly used human or mouse reference genes. A perfect product for finding the most optimal reference gene for
your samples. A one year license of GenEx Standard software with GeNorm and
Normfinder is also included in the kit.
VisiBlue™ qPCR mix colorant
The VisiBlue mastermix colorant enables you to color your favourite qPCR mastermix to easily visualize where the reagent is loaded to your plates and tubes.
VisiBlue is very easy to use by simple addition to your favorite master mix.
CelluLyser™ - for rapid and easy lysis and cDNA synthesis
The CelluLyser™ Lysis and cDNA Synthesis Kit enables you to generate cDNA
from small samples with minimal losses and hands-on time. It is particularly
useful for single cell analysis. By using CelluLyser™, the entire workflow from
cell lysis to RT and qPCR can be performed without washing steps, thus eliminating material loss.
TATAA Interplate Calibrator - Variation Compensation
For practical reasons many qPCR studies involve the use of samples that are
processed in more than a single batch or in which the sample set is extended
over time. Even over a short time period, variation between qPCR processing
runs is observed due to different baseline subtractions and threshold settings.
The TATAA Interplate Calibrator (IPC) is used to compensate for the variation
between qPCR runs.
14
Express your
genius
TATAA Biocenter, with offices in
Gothenburg, San Francisco and
Prague is the leading provider of
real-time PCR services and the prime
organizer of real-time PCR workshops globally. TATAA Biocenter conducts commissioned research and
training within the field of molecu-
lar diagnostics and gene expression
analysis, along with developing realtime PCR expression panels. TATAA
Biocenter has great experience and
expertise in high resolution gene
expression profiling, pathogen detection, and small sample/single cell
analysis.
TATAA Biocenter AB
Odinsgatan 28, 411 03 Göteborg
Tel: +46 31 761 57 00, Fax: +46 31 15 28 90
E-mail: [email protected], Website: www.tataa.com